The overall objectives of the proposed research are to understand the molecular mechanisms by which damage induced in DNA by alkylating agents is repaired. We would like to understand the way in which mutations are induced by alkylating agents in eucaryotic organisms. EMS and MNNG, which are both potent mutagens and carcinogens, will be used as model alkylating agents in the yeast Saccharmyces cerevisiae. We will study the molecular mechanisms and genetic control of alkylation mutagenesis using specific mutations in the CYC1 gene as well as other genetic markers. EMS and MNNG induced reversion of the various markers will be examined in repair proficient and repair deficient yeast strains. We will determine whether EMS and MNNG show differential mutability of various markers at different stages during the mitotic cell cycle and whether the effect is a property of only certain alleles. Experiments on whether there is an adaptive response for alkylation damage and repair in yeast and its genetic control will be carried out. In addition, we will determine which epistatic group(s) exist for repair of alkylation damage in both haploid and diploid yeast. We will also characterize and describe the repair processes occurring for alkylation damage. This work will involve the following: quantitative analysis of products formed in yeast nuclear DNA following treatment of cells with EMS and MNNG; studies on the ability of wild type and mutant yeast to remove O6 alkyl guanine from nuclear DNA; characterization of apurinic endonucleases in wild type and mutant yeast strains; and ability of wild type and mutant yeast strains to repair alkylation-induced single strand breaks in DNA.